Approximately 80% of chronic pancreatitis cases are associated with alcohol consumption. Analogous to liver fibrosis and hepatic stellate cells, there is increasing evidence for a key role in the activation of the pancreatic stellate cell in pancreatitis. As in the liver, the central gateway for this process may be NADPH oxidase. When the reactive oxygen species generated by NADPH oxidase overwhelm the innate anti-oxidant system in cells, then tissue injury can result. In susceptible individuals these injuries can lead to chronic activation of the inflammatory response and ultimately pancreatitis. Although much is known about the composition and activation of the NADPH oxidase system in neutrophils, little is known about the complex in other tissues. For example, this proposal contains the first report of the presence of components of this reactive oxygen species generating complex in activated pancreatic stellate cells. Current knowledge is limited by the available antibodies that target NADPH oxidase subunits, and mRNA experiments that show the presence of protein precursors. Because of these limitations and the importance of this system in alcoholic pancreatitis, we propose to identify the components of the NADPH oxidase complex from pancreatic stellate cells by state-of-the-art methods. The specific hypothesis we will be testing is that alcohol and growth factors alter the subunit composition of the NADPH oxidase system in pancreatic stellate cells, leading to a synergistic increase in the activity of the NADPH oxidase, an effect that mediates several pathologic responses in the pancreas. Pancreatic stellate cell cultures will be prepared from rat pancreas by established methods and treated with PDGF and ethanol, alone and in combination. Membrane fractions will be isolated from these cultures and analyzed by blue native two-dimensional gels. For the first dimension electrophoretic separation will be used with a non-denaturing buffer that enables separation of complexes. The second dimension utilizes SDS-PAGE denaturing conditions to separate the component parts of each complex isolated in the first dimension of the gel. In-gel tryptic digestion followed by microbore liquid chromatography coupled to tandem mass spectrometry with data dependant acquisition will be used to collect mass spectra and tandem mass spectra of the digest components. Data sets will be screened against human genomic databases of predicted or known open reading frame translations. Significant matches will be examined manually to confirm assignments. In this way components of the complex will be identified by mass and sequence information derived from in-gel digests. These data are essential for the successful development of targeted therapeutic agents for treatment of alcohol related pancreatitis. By far the most common cause of chronic pancreatitis is alcohol abuse, estimated to cause 70- 90% of the cases. The processes of chronic pancreatitis include inflammation and scarring with the loss of tissue. Of particular note, the processes of this condition continue even after cessation of alcohol intake, indicating that the processes are self-sustaining once a certain stage of the disease is reached. Adding to the morbidity and mortality of this disorder is the fact that patients with chronic pancreatitis are at a significantly increased risk for pancreatic cancer. Successful completion of the experiments described in this proposal are essential for the development of targeted therapeutic agents for treatment of alcohol related pancreatitis. [unreadable] [unreadable] [unreadable]